Measuring dynamic guanosine signaling at the site of focal ischemia over time remains challenging to probe with existing technology yet knowledge of the dynamics, regulatory mechanisms, and function of local guanosine fluctuations during ischemia would positively impact our understanding of the brain?s immediate local neuroprotective response. Guanosine is a nucleoside purine which has been postulated to play a potent restorative role after ischemic injury; however, to date, the mechanism and dynamics of guanosine action in the brain remains unresolved. Additionally, knowledge of the extent to which guanosine signaling changes as a function of ischemia duration and severity would provide critical insight into guanosine?s role as a neuroprotector. We propose to solve a significant gap in the understanding of guanosine signaling dynamics during focal ischemia by developing a microfluidic platform to initiate sustained local oxygen-glucose deprivation in a sub-region of a brain slice and using fast-scan cyclic voltammetry (FSCV) recording of guanosine with millisecond-to-second temporal resolution to provide critical insight into the mechanisms of guanosine regulation. Measuring local guanosine dynamics at the site of injury with significantly improved spatiotemporal resolution will provide critical information of the brain?s immediate local damage response. This proposal fits within our long-term goal to develop analytical tools to detect and understand dynamic neurochemical-regulated inflammation in the brain during injury. The rationale for this proposal is that these tools will provide knowledge of the dynamics, mechanism, and function of rapid guanosine signaling in the brain during ischemia for the first time which could further inform the development of guanosine- targeted therapies for neurological injury. The proposal will be completed by the following three specific aims: (1) Develop microfluidic platforms for delivery of spatiotemporally controlled and sustained focal ischemia to brain slices, (2) Characterize the mechanism of rapid guanosine release and clearance in the hippocampus as a function of ischemia severity and location, and (3) Characterize the impact of rapid guanosine signaling on local adenosine changes during focal ischemia. We will pursue these aims with an innovative approach by using novel microfluidic platforms for time-controlled delivery of ischemia to brain slices coupled to rapid electrochemical recording of guanosine signaling with FSCV for the first time. This work is significant because these studies will enable extraordinary mechanistic insight into the brain?s immediate response to ischemia over varying ischemia durations and severities which will directly impact future therapeutic strategies for brain injury. The tools are translatable to any biological system to study local tissue responses. The expected outcome is a new platform to investigate rapid endogenous guanosine signaling in the brain for the first time and an in-depth understanding of guanosine regulation and neuromodulation during ischemia. This work will have a positive impact on how guanosine is studied and will significantly advance knowledge of guanosine?s role in the brains immediate damage response.
PROJECT NARATIVE The proposed research is relevant to public health because understanding the dynamics and regulatory mechanisms of guanosine signaling in the brain during ischemia is expected to advance knowledge of the brains immediate neuroprotection response and provide critical information on potential therapeutic targets for ischemia. Methods to monitor rapid guanosine signaling at the local site of ischemia during injury are lacking and we will bridge this critical gap by developing new platforms which combine millisecond-to-second guanosine recording with microfluidic focal brain stimulation. Thus, this proposal is relevant to part of the NIH?s mission that pertains to fostering fundamental creative discoveries and innovative research strategies as a basis for protecting and improving health.